Nanoscale Metal–Organic Particles with Rapid Clearance for

Nanoscale Metal−Organic Particles with Rapid Clearance for Magnetic Resonance ImagingGuided Photothermal Therapy Yu Yang,† Jingjing Liu,‡ Chao Liang,‡ Liangzhu Feng,‡ Tingting Fu,§ Ziliang Dong,‡ Yu Chao,‡ Yonggang Li,§ Guang Lu,*,‡ Meiwan Chen,*,† and Zhuang Liu*,‡ †

State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Avenida da Universidad, Taipa, Macau, China ‡ Institute of Functional Nano & Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Soochow University, Suzhou, Jiangsu 215123, China § Department of Radiology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, China S Supporting Information *

ABSTRACT: Nanoscale metal−organic particles (NMOPs) are constructed from metal ions and organic bridging ligands via the self-assembly process. Herein, we fabricate NMOPs composed of Mn2+ and a near-infrared (NIR) dye, IR825, obtaining Mn-IR825 NMOPs, which are then coated with a shell of polydopamine (PDA) and further functionalized with polyethylene glycol (PEG). While Mn2+ in such Mn-IR825@PDA−PEG NMOPs offers strong contrast in T1-weighted magnetic resonance (MR) imaging, IR825 with strong NIR optical absorbance shows efficient photothermal conversion with great photostability in the NMOP structure. Upon intravenous injection, Mn-IR825@PDA−PEG shows efficient tumor homing together with rapid renal excretion behaviors, as revealed by MR imaging and confirmed by biodistribution measurement. Notably, when irradiated with an 808 nm laser, tumors on mice with Mn-IR825@PDA−PEG injection are completely eliminated without recurrence within 60 days, demonstrating the high efficacy of photothermal therapy with this agent. This study demonstrates the use of NMOPs as a potential photothermal agent, which features excellent tumortargeted imaging and therapeutic functions, together with rapid renal excretion behavior, the latter of which would be particularly important for future clinical translation of nanomedicine. KEYWORDS: nanoscale metal−organic particles, photothermal therapy, MR imaging, body clearance

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ability and potential long-term toxicity of many inorganic nanoparticles have remarkably impeded their progress for future clinical translation. On the other hand, the easy photobleaching of many small organic dyes, such as the clinically approved NIR dye indocyanine green (ICG), has also limited their further applications. Therefore, the development of photothermal agents with strong NIR absorbance, excellent photostability, imaging capability to allow real-time tracking, as well as rapid clearance properties remains an important goal to achieve the development of cancer PTT technology.

hotothermal therapy (PTT), which utilizes heat generated from photothermal agents under near-infrared (NIR) light irradiation to ablate cancerous cells, has been extensively explored and found to be a promising alternative approach for future cancer treatment.1−7 During PTT, imaging is often needed to identify the tumor location and morphology for a precise tumor ablation, as well as to monitor the tumor accumulation of photothermal agents to allow irradiation at the best timing. Until now, a large number of inorganic and organic photothermal agents, including various gold nanostructures,8−15 carbon nanomaterials,16 transitionmetal chalcogenides,17−21 conjugated polymers,22−24 and small organic dyes,25−28 have been widely explored for efficient photothermal therapy of cancers. However, the non-biodegrad© 2016 American Chemical Society

Received: December 14, 2015 Accepted: January 22, 2016 Published: January 22, 2016 2774

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ACS Nano Metal−organic frameworks (MOFs) as well as coordination polymers, the metal−organic solid materials consisting of metal ions or clusters linked by organic bridging ligands, have received tremendous attention in recent years from many different areas.29−38 In the field of biomedicine, in particular, nanoscale metal−organic particles (NMOPs) designed with judicious selection of inorganic and organic building blocks have shown great potential as a multifunctional nanoplatform for carrying imaging agents,36 gene therapeutics,39,40 chemotherapeutics,41 and photosensitizers,42 mostly aiming at applications in biomedical imaging and cancer therapy. One important feature of NMOPs is that, unlike typical inorganic nanoparticles or organic conjugated polymers, NMOPs are formed based on a noncovalent bond and thus may be gradually decomposed via ligand exchange reactions with monodentate ligands (for example, phosphate) and/or water molecules in biological systems.43 However, although in a recent work, a MOF has been used to encapsulate Fe3O4 nanocrystals to obtain composite nanoparticles for photothermal cancer treatment by employing the NIR absorbance of Fe3O4,44 and the applications of NMOPs or other MOF-related nanostructures by themselves as photothermal agents have not yet been reported to the best of our knowledge. Herein, we design a new type of NMOP via self-assembling of Mn2+ ions and IR825 bridging ligands, which offer strong MR contrast and exhibit high NIR absorbance, respectively, and for the first time demonstrate the use of NMOPs as a trackable, biodegradable, and highly effective agent for imaging-guided photothermal cancer treatment. The obtained Mn-IR825 NMOPs show an r1 relaxivity comparable or even higher than that of a commercially used Gd-based contrast agent, as well as an NIR mass extinction coefficient much larger than that of the most commonly explored inorganic photothermal agents and conjugated polymers. Those NMOPs are then coated with polydopamine (PDA) to facilitate further modification with polyethylene glycol (PEG), which confer nanoparticles excellent physiological stability. As observed by MR imaging and further confirmed by quantitative biodistribution measurement, Mn-IR825@PDA−PEG NMOPs after intravenous (i.v.) injection showed efficient tumor accumulation and, in the meanwhile, could be rapidly excreted via the kidneys into urine. In vivo photothermal ablation therapy is further realized in a mouse tumor model with i.v.-injected Mn-IR825@PDA−PEG, demonstrating an excellent tumor elimination effect upon NIR irradiation of tumors. NMOPs developed here may thus be a new class of photothermal agents with biodegradability to allow rapid excretion and minimize long-term toxicity, an imaging function to facilitate real-time tracking, as well as great photothermal performances such as a rather high mass extinction coefficient together with robust photothermal stability.

Figure 1. Preparation and characterization of Mn-IR825@PDA− PEG. (a) Schematic illustration to show the fabrication process of Mn-IR825@PDA−PEG. (b,c) TEM images of Mn-IR825 (b) and Mn-IR825@PDA−PEG (c). (d) UV−vis−NIR spectra of IR825 in methanol and Mn-IR825@PDA−PEG in water. (e) Temperature curves of ICG and Mn-IR825@PDA−PEG solutions under five cycles of photothermal heating by an 808 nm laser.

Subsequently, dopamine was mixed with the yielded Mn-IR825 NMOPs at pH 8.5 to form a thin layer of PDA on the surface of NMOPs, which were further conjugated with amine-terminated PEG under an alkaline pH via a Michael addition reaction. The obtained NMOPs are stable in common organic solvents (such as DMF and methanol). Powder X-ray diffraction (XRD) measurements indicated the amorphous nature of Mn-IR825 NMOPs. Transmission electron microscopy (TEM) images showed the average diameter of as-made Mn-IR825 NMOPs to be ∼40 nm (Figure 1b). After being coated with PDA and PEG, the average size of Mn-IR825@PDA−PEG showed a slight increase (Figure 1c). Dynamic light scattering (DLS) data illustrated the average hydrodynamic size of Mn-IR825@PDA− PEG to be ∼70 nm (Supporting Information Figure S1), which was a little bigger than that observed under TEM. Meanwhile, the ζ-potential of Mn-IR825@PDA−PEG was measured to be −15.0 ± 1.8 mV. Similar to IR825 in methanol, both Mn-IR825 NMOPs and Mn-IR825@PDA−PEG exhibited a strong NIR absorption peak at 825 nm (Figure 1d). The mass extinction coefficient of IR825 in NMOPs was calculated to be 78.2 L/g/cm at 808 nm, which was much higher than that of many previously reported photothermal agents (e.g., 28.4 L/g/cm for MoS2 nanosheets, 46.5 L/g/cm for single-walled carbon nanotubes, 62 L/g/cm for polypyrrole).4,18,45 Note that the NIR absorbance contributed by PDA in our Mn-IR825@PDA−PEG nano-

RESULTS AND DISCUSSION Mn-IR825@PDA−PEG NMOPs were fabricated according to the following procedure illustrated in Figure 1a. IR825 dissolved in methanol was added into a methanol/N,Ndimethylformamide (DMF) (85/15 v/v) solution containing MnCl2 under sonication for 6 h followed by an additional 12 h of stirring at 30 °C. Then, poly(vinylpyrrolidone) (PVP, 30 kDa) as a dispersing and stabilizing reagent was added into the reaction mixture to control the size of NMOPs (large aggregates would appear in the absence of PVP). After being stirred for another 60 h, Mn-IR825 NMOPs were obtained. 2775

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Figure 2. In vitro experiments. (a−c) Relative viabilities of 293T cells (a), HeLa cells (b) and A549 cells (c) incubated with Mn-IR825@PDA− PEG at various concentrations for 24 and 48 h. (d) Relative viabilities of 4T1 cells incubated with various concentrations of Mn-IR825@ PDA−PEG with and without the 808 nm laser irradiation (0.5 W/cm2) for 5 min. (e) Confocal fluorescence images of Mn-IR825@PDA− PEG-incubated 4T1 cancer cells after irradiation by the 808 nm laser at different power densities for 5 min. The cells were co-stained by calcein AM and propidium iodide before imaging. Scale bar: 150 μm.

particles with only a thin layer of PDA coating could almost be ignored. Furthermore, NMOPs showed efficient photothermal conversion with excellent photostability exposed to an 808 nm NIR laser (Figure 1e and Figure S2). In contrast, although ICG, a widely used NIR dye, also showed great photothermal heating performance in the first round of NIR laser irradiation, it would quickly lose its NIR absorbance due to the photobleaching effect after several cycles of NIR laser exposure (Figure 1e and Figure S3). Before exploring its in vivo performance, we evaluated the biocompatibility of Mn-IR825@PDA−PEG using the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Our results demonstrated that Mn-IR825@ PDA−PEG did not show any obvious toxic effects to 293T cells (a human embryonic kidney cell line), A549 cells (a human alveolar basal epithelial cell line), HeLa cells (a human epithelioid cervix carcinoma cell line), and 4T1 cells (a murine breast cancer cell line) after incubation for 24 or 48 h even with a high concentration up to 200 mg/mL, indicating the excellent biocompatibility of Mn-IR825@PDA−PEG (Figure 2a−c and Figure S4). Next, various concentrations of Mn-IR825@PDA− PEG as photothermal agents were added into the 4T1 cancer cells for in vitro cancer cell killing upon exposure to the 808 nm NIR laser. As expected, MTT results showed that 4T1 cells were killed in a concetration-dependent manner after NIRinduced PTT with Mn-IR825@PDA−PEG (Figure 2d). Meanwhile, confocal fluorescence imaging of calcein AM and propidium (PI) co-stained 4T1 cells also demonstrated the high PTT ablation efficacy by Mn-IR825@PDA−PEG (Figure 2e). Furthermore, flow-cytometry-based apoptosis analysis of annexinV-FITC and PI double-stained 4T1 cells showed that

the major PTT-induced cell death type was necrosis (Figure S5). Mn2+ with five unpaired 3d electrons has been demonstrated to be an effective T1 contrast agent in MR imaging (Figure 3a). The T1-weighted MR images of Mn-IR825@PDA−PEG solutions showed a concentration-dependent brightening effect under a 3 T MR clinical scanner. The corresponding longitudinal relaxivity (r1) value of Mn-IR825@PDA−PEG was calculated to be 7.48 ms−1 s−1, which is higher than that of a commercially used Gd-based contrast agent, Magnevist (4.25 mM−1 s−1).46 Upon i.v. injection of Mn-IR825@PDA−PEG at the dose of 5 mg/kg (in terms of IR825), T1-weighted MR signals gradually showed up in the tumor, indicating timedependent tumor accumulation of NMOPs (Figure 3c). Interestingly, the mouse kidneys also showed up with strong T1 signals at different times post-injection (p.i.) of Mn-IR825@ PDA−PEG under MR imaging, indicating significant renal filtration of Mn2+ (Figure 3d). Quantitative analysis further confirmed that the average MR signals in the tumor, kidneys, and liver of mice post-injection of NMOPs all gradually increased over time (Figure 3e,f). To further verify our MR imaging results, we then studied the in vivo behaviors of Mn-IR825@PDA−PEG by measuring Mn2+ levels in various organs using inductively coupled plasma atomic emission spectroscopy (ICP-AES). The blood levels of Mn-IR825@PDA−PEG reduced gradually over time but were maintained at a relatively high level even at 24 h p.i. (Figure 4a). The first and second phase blood circulation half-lives for those nanoparticles were determined to be 0.65 ± 0.18 and 5.01 ± 1.23 h, respectively. Meanwhile, as presented in Figure 4b, Mn-IR825@PDA−PEG showed a rather high tumor 2776

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Figure 3. In vivo MR imaging of Mn-IR825@PDA−PEG. (a,b) T1-weighted MR images and T1 relaxation rates (r1) of Mn-IR825@PDA−PGE solutions at various Mn2+ concentrations. (c,d) T1-weighted MR images of 4T1 tumor-bearing mice at different time post-injection of MnIR825@PDA−PEG injection. The tumors, livers, and kidneys are highlighted by red arrows, yellow arrows, and red circles, respectively. (e,f) Quantification analysis of T1-weighted MR signals in different organs.

accumulation at ∼8% of injected dose per gram tissue (%ID/g) at 24 h p.i., consistent with the MR imaging results. The efficient tumor retention of our nanoparticles should be attributed to the enhanced permeability and retention effect of cancerous tumors, whose leaky and tortuous vasculatures are favorable for passive accumulation of nanoparticles, particularly those with long blood circulation time. To further understand the in vivo excretion kinetics, tumorfree Balb/c mice after i.v. injection of Mn-IR825@PDA−PEG were sacrificed at 1 day, 3 days, and 7 days p.i., with Mn2+ concentrations in various organs analyzed using ICP-AES (Figure 4c). The accumulation of Mn-IR825@PDA−PEG in liver and spleen was determined to be ∼12 and ∼8%ID/g at 1 day p.i. and further decreased to rather low levels later on. Compared to many other inorganic nanoparticles, the quantity of Mn2+ retention in the mouse body after 7 days could be much lower. Notably, kidneys showed rather high Mn2+ signals just as observed by MR imaging, indicating that IR825@PDA− PEG might be quickly removed from the body via kidneys. Next, the Mn2+ concentrations in feces and urine collected from Mn-IR825@PDA−PEG-injected mice were also analyzed using

ICP-AES. It was found that 31.23 and 8.34%ID of injected Mn were cleared out via urine and feces, respectively, within the first 12 h (Figure 4d). High levels of Mn2+ were detected mainly in the urine of injected mice in the following days. Our results thus evidenced that Mn-IR825@PDA−PEG nanoparticles could be rapidly excreted, mainly via the renal excretion pathway. This is likely due to the intrinsic biodegradability of the NMOP structure, which is constructed by the noncovalent coordination interaction.47 Due to the high affinity between IR825 and albumin, we did observe a small amount of IR825 release from NMOP nanoparticles after incubation in serum-containing medium for 12 h (Figure S6). Therefore, Mn-IR825 NMOPs may be gradually degraded into free organic molecules and metal ions in the complicated in vivo environment and get excreted afterward. The efficient tumor retention together with rapid renal clearance behaviors of such NMOPs is particularly promising for effective and safe cancertargeted therapy. Motivated by the excellent in vitro photothermal therapy efficiency of Mn-IR825@PDA−PEG and its high tumor accumulation, we evaluated the in vivo PTT efficiency of Mn2777

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Figure 4. In vivo behaviors of Mn-IR825@PDA−PEG as determined by measuring Mn2+ levels with ICP-AES. (a) Blood circulation of MnIR825@PDA−PEG. The pharmacokinetics of Mn-IR825@PDA−PEG followed the two-compartment model. (b) Biodistribution of MnIR825@PDA−PEG in 4T1-tumor-bearing mice at 24 h p.i. (c,d) Time-dependent biodistribution (c) and excretion profiles (d) of Mn2+ in healthy mice after i.v. injection of Mn-IR825@PDA−PEG.

Figure 5. In vivo photothermal therapy. (amb) IR thermal images (a) and tumor temperature changes (b) of 4T1 tumor-bearing mice i.v.injected with Mn-IR825@PDA−PEG (5 mg/kg) or PBS. At 24 h p.i., their tumors were irradiated under the 808 nm laser at the intensity of 0.6 W/cm2. (c) Growth curves of 4T1 tumors on mice of different groups after corresponding treatments as indicated (n = 5). The relative tumor volumes were normalized to their initial sizes. (d) Survival curves of various groups of mice after different treatments. (e) H&E-stained images of major organs from healthy control mice and Mn-IR825@PDA−PEG-injected mice 60 days after PPT treatment. Scale bar: 200 μm.

IR825@PDA−PEG on 4T1 tumor-bearing mice. First, the in vivo photothermal heating profiles of tumors were recorded by an IR thermal camera (Figure 5a). It was found that the

temperature of tumors on mice 24 h after i.v. injection with Mn-IR825@PDA−PEG (5 mg/kg, in terms of IR825) would rapidly rise from ∼34 to ∼52 °C within 5 min under the 2778

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ACS Nano irradiation of an 808 nm laser at 0.6 W/cm2, whereas tumors on a phosphate-buffered saline (PBS)-injected mouse showed only ∼4 °C increase under the same irradiation (Figure 5b). Then the tumor sizes in different groups of mice were monitored (Figure 5c). It was found that tumors in the PTT group with Mn-IR825@PDA−PEG injection and NIR laser irradiation were completely eliminated without recurrence during 18 days. In marked contrast, tumors in other control groups, including PBS injection without or with laser irradiation, as well as NMOP injection without laser exposure, all showed rapid growth (Figure 5c and Figure S7). Notably, mice after PTT treatment were tumor-free and survived for more than 60 days, while the average life spans of mice in the other three control groups were less than 24 days, strongly suggesting Mn-IR825@ PDA−PEG to be an efficient agent for in vivo photothermal ablation of tumors (Figure 5d). Furthermore, we found that mice after injection with our NMOPs and PTT treatment behaved normally without a noticeable decrease in body weight (Figure S8). No appreciable sign of organ damage or inflammatory lesion was noticed for mice 60 days after NMOP-based PTT therapy, as revealed by hematoxylin and eosin (H&E)-stained major organ slices of those mice (Figure 5e). Our previous study has uncovered that IR825 could be excreted from the mouse body without longterm toxicity.26 On the other hand, Mn2+ has been reported to be a relatively safe metal ion with low toxicity and rapid clearance.48−50 Therefore, our Mn-IR825@PDA−PEG, which could be gradually degraded in vivo and showed rapid clearance, would be a safe agent for in vivo use.

Synthesis of Mn-IR825@PDA−PEG. IR825, a heptamethine indocyanine dye was synthesized according to the previous literature.26,51 The Mn-IR825 NMOPs were prepared using IR825 as the organic ligand, manganese ions as metallic nodes, PVP as dispersing and stabilizing reagent, and methanol/DMF (85/15 v/v) as the solvent. In a typical synthesis experiment, 2.8 mg of IR825 was dissolved in a methanol−triethylamine (50:1) mixture and then added into a 20 mL methanol/DMF (85/15 v/v) solution of MnCl2. After ultrasonication for 6 h and stirring for 12 h at 30 °C, 10 mg of PVP (MW = 30 K) was used as dispersing and stabilizing agent. After being further stirred for 60 h, Mn-IR825 nanoscale coordination polymer nanoparticles (NMOPs) were obtained. For coating with PDA, 4 mg of dopamine was added and mixed with the synthesized Mn-IR825 NMOPs under pH 8.5. After being stirred for 4 h, excess unreacted dopamine molecules were removed by ultrafiltration using 100 kDa MWCO filters. Next, another 40 mg of six-arm PEG-amine (10 kDa) and 40 mg of mPEG-amine (5 kDa) were added into the Mn-IR825@ PDA, whose pH was adjusted to 9.0 under ultrasonication for 30 min and stirring for 12 h. After purification by ultrafiltration, the concentration of Mn-IR825@PDA−PEG was determined by IR825. Cellular Experiment. HeLa human cervical cancer cells, A549 human alveolar basal epithelial cells, and 4T1 murine breast cancer cells were originally obtained from American Type Culture Collection (ATCC) and cultured in standard cell media recommended by ATCC with 10% fetal bovine serum and 1% penicillin/streptomycin. For in vitro cytotoxicity assay, HeLa, A549, and 4T1 cells were seeded into 96-well plates at 1 × 105/well until adherent and then incubated with various concentrations of Mn-IR825@PDA−PEG for 24 and 48 h. The standard MTT assays were measured to determine the relative cell activities. For in vitro photothermal therapy, 4T1 cancer cells were seeded into 96-well plates at 1 × 105/well until adherent and then incubated with various concentrations of Mn-IR825@PDA−PEG at 37 °C. Then the cells were exposed to an 808 nm NIR laser at the power density of 0.5 W/cm2 for 5 min. The relative cell viabilities after photothermal ablation were measured using the standard MTT assay. For confocal imaging, 4T1 cancer cells after irradiation by the 808 nm laser at the power density of 0.5 W/cm2 for 5 min were stained by calcein AM and PI and then imaged using a Leica SP5 laser scanning confocal microscope. Tumor Model. Balb/c mice were obtained from Nanjing Pengsheng Biological Technology Co. Ltd. and used under the protocol approved by Soochow University Laboratory Animal Center. For the 4T1 tumor model, 2 × 106 cells in 40 μL of serum-free RMPI1640 medium were injected onto the back of each Balb/c mouse. In Vivo MR Imaging. Mn-IR825@PDA−PEG with various concentrations (0−0.22 mM) was scanned under a 3 T clinical MRI scanner at room temperature. The T1-weighted MR signal intensities were acquired from MR images via manually drawn regions of interest. Then the relaxation rate r1 was calculated as the reciprocal of T1 (r1 = 1/T1) at various Mn2+ concentrations. For in vivo MR imaging, Balb/c mice inoculated with 4T1 murine breast cancer tumors were i.v.injected with Mn-IR825@PDA−PEG (5 mg/kg for each mouse) when the tumor size reached ∼50 mm3. Small animal MR images were collected and analyzed on a 3 T clinical MRI scanner equipped with a special animal imaging coil. Blood Circulation, Biodistribution, and Clearance. For blood circulation of Mn-IR825@PDA−PEG, blood was collected from the each mouse at the indicated time points, weighed, and then dissolved in digesting chloroazotic acid (HNO3/HCl = 3:1) to analyze the total amount of Mn in the blood using ICP-AES. To determine the biodistribution of our nanoparticles, major organs and tissues (liver, spleen, kidney, heart, lung, stomach, intestine, skin, muscle, bone, and tumor) from Balb/c mice (n = 3) were collected at the indicated time point. Next, the collected organs were wet-weighted and solubilized in chloroazotic acid under heating to boiling for 2 h. After each sample was diluted with deionized water to 10 mL, ICP-AES was used to measure Mn2+ concentrations in different samples. To study the excretion of nanoparticles, mice after i.v. injection with Mn-IR825@ PDA−PEG were housed in metabolic cages to collect their urine and

CONCLUSION In summary, we for the first time developed NMOPs for imaging-guided photothermal therapy of cancer. Such NMOPs are fabricated by simply mixing IR825 dye with Mn2+, which would self-assemble into nanoparticles under a mild temperature. The surface coating of those NMOPs, by coating with a PDA shell and further PEGylation, is also a rather simple and effective method that may be extended to the surface modification of various types of coordination polymer-based nanostructures. The obtained IR825@PDA−PEG NMOPs are featured with a number of unique advantages over commonly explored photothermal agents: (1) The photothermal and MR imaging agents are self-assembled to form a “carrier-free system” with high loading efficiencies; (2) IR825 bridging ligand exhibits extremely high NIR absorption and excellent photothermal stability; (3) Mn2+ as the connecting ions in those NMOPs offers strong T1 MR contrast, which would be useful for tumor imaging and the planning of PTT therapy; (4) those NMOPs show rapid renal excretion, which would minimize the long-term toxicity concerns of those nanoparticles, showing promise for future clinical translation. With efficient passive tumor homing, effective photothermal ablation of tumors with those NMOPs is then achieved in our mouse tumor model experiment. Our work thus demonstrates the great potential of MNOPs, or other MOF-like nanostructures, in general, for applications in nanomedicine and cancer theranostics. EXPERIMENTAL SECTION Materials. MnCl2, dopamine, and ICG were bought from SigmaAldrich. Polylactide−poly(ethylene glycol) was purchased from Biomatrik Inc. Other chemicals were obtained from Sinopharm Group Co. Ltd. 2779

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ACS Nano feces. The collected urine and feces were digested by chloroazotic acid and measured by ICP-AES. In Vivo Photothermal Therapy. Balb/c mice bearing 4T1 tumors were divided into four groups when the tumors reached ∼60 cm3 (n = 5 for each group): (a) untreated control, (b) i.v. injection with MnIR825@PDA−PEG (200 μL, 0.5 mg/mL in terms of IR825), (c) irradiated with NIR laser (808 nm, 0.6 W/cm2, 5 min), and (d) i.v. injection with Mn-IR825@PDA−PEG and irradiated under NIR laser. The tumor temperature changes of mice were monitored by an IRS E50 Pro Thermal imaging camera. The tumor sizes were measured every other day with a digital caliper and calculated as volume = wildth2 × length/2. The relative tumor volume was analyzed according to the formula V/V0 (where V0 is the tumor volume when the treatment was initiated). Tissue Slicing and Staining. For histology analysis, three mice from PTT treatment groups 2 months post-injection of Mn-IR825@ PDA−PEG (5 mg/kg) and three age-matched healthy mice as the control were sacrificed. The harvested organs were fixed with 10% neutral buffered formalin, treated routinely into paraffin, and sectioned at 8 μm thickness. Slices of those organs including liver, kidney, spleen, heart, lung, and intestine were stained with H&E and observed by a digital microscope (Leica QWin).

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ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.5b07882. Additional characterization data, relative cells viabilities, flow-cytometry-based apoptosis, and body weights of mice after different treatments (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was partially supported by the National Natural Science Foundation of China (51525203), the National “973” Program of China (2012CB932601), a Jiangsu Natural Science Fund for Distinguished Young Scholars, the Macao Science and Technology Development Fund (062/2013/A2), the Research Fund of the University of Macau (MYRG2014-00033-ICMSQRCM, MRG004/CMW/2014/ICMS), and the Collaborative Innovation Center of Suzhou Nano Science and Technology. REFERENCES (1) Song, G.; Wang, Q.; Wang, Y.; Lv, G.; Li, C.; Zou, R.; Chen, Z.; Qin, Z.; Huo, K.; Hu, R.; et al. A Low-Toxic Multifunctional Nanoplatform Based on Cu9S5@mSiO2 Core-Shell Nanocomposites: Combining Photothermal and Chemotherapies with Infrared Thermal Imaging for Cancer Treatment. Adv. Funct. Mater. 2013, 23, 4281− 4292. (2) Cheng, L.; Wang, C.; Feng, L.; Yang, K.; Liu, Z. Functional Nanomaterials for Phototherapies of Cancer. Chem. Rev. 2014, 114, 10869−10939. (3) Huang, X.; El-Sayed, I. H.; Qian, W.; El-Sayed, M. A. Cancer Cell Imaging and Photothermal Therapy in the Near-infrared Region by Using Gold Nanorods. J. Am. Chem. Soc. 2006, 128, 2115−2120. (4) Robinson, J. T.; Tabakman, S. M.; Liang, Y.; Wang, H.; Sanchez Casalongue, H.; Vinh, D.; Dai, H. Ultrasmall Reduced Graphene 2780

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